2.9 Aortic ring assays
To establish the direct action of FGF2 and PEG-FGF2 conjugates on vascular tissue, the thoracic aortae of 8-week-old mice from each group were isolated surgically, cleaned, and dissected into 0.5 mm rings. Rings were then embedded in 1 mg/mL of type I collagen (Millipore, Cat#08-115) in a 96-well plate as described previously (Baker et al. , 2011). The embedded rings were cultured in HG (33 mM) serum-free endothelial basal medium (EBM) (Lonza, Cat#CC-3121) in the presence of FGF2 or PEG-FGF2 conjugates. During the exponential growth phase, angiogenic response data were obtained by counting the endothelial microvessel sprouts growing out from the cultured rings. Before the regression phase, rings were fixed with 4% (w/v) paraformaldehyde for 30 min. Pictures were taken on day 12 with a computer assisted microscope (Eclipse Ni, NIKON), and the total number of branches was counted using ImageJ (National Institutes of Health, USA).